Therapies using mesenchymal stem cell (MSC) seeded scaffolds may be applicable to various fields of regenerative medicine, including craniomaxillofacial surgery. Plastic compression of collagen scaffolds seeded with MSC has been shown to enhance the osteogenic differentiation of MSC as it increases the collagen fibrillary density. The aim of the present study was to evaluate the osteogenic effects of dense collagen gel scaffolds seeded with mesenchymal dental pulp stem cells (DPSC) on bone regeneration in a rat critical-size calvarial defect model. Two symmetrical full-thickness defects were created (5 mm diameter) and filled with either a rat DPSC-containing dense collagen gel scaffold (n = 15), or an acellular scaffold (n = 15). Animals were imaged in vivo by microcomputer tomography (Micro-CT) once a week during 5 weeks, whereas some animals were sacrificed each week for histology and histomorphometry analysis. Bone mineral density and bone micro-architectural parameters were significantly increased when DPSC-seeded scaffolds were used. Histological and histomorphometrical data also revealed significant increases in fibrous connective and mineralized tissue volume when DPSC-seeded scaffolds were used, associated with expression of type I collagen, osteoblast-associated alkaline phosphatase and osteoclastic-related tartrate-resistant acid phosphatase. Results demonstrate the potential of DPSC-loaded-dense collagen gel scaffolds to benefit of bone healing process.
Stem cells endowed with skeletogenic potentials seeded in specific scaffolds are considered attractive tissue engineering strategies for treating large bone defects. In the context of craniofacial bone, mesenchymal stromal/stem cells derived from the dental pulp (DPSCs) have demonstrated significant osteogenic properties. Their neural crest embryonic origin further makes them a potential accessible therapeutic tool to repair craniofacial bone. The stem cells' direct involvement in the repair process versus a paracrine effect is however still discussed. To clarify this question, we have followed the fate of fluorescent murine DPSCs derived from PN3 Wnt1-CRE-Rosa Tomato mouse molar (T-mDPSCs) during the repair process of calvaria bone defects. Two symmetrical critical defects created on each parietal region were filled with (a) dense collagen scaffolds seeded with T-mDPSCs, (b) noncellularized scaffolds, or (c) no scaffold. Mice were imaged over a 3-month period by microcomputed tomography to evaluate the extent of repair and by biphotonic microscopy to track T-mDPSCs. Histological and immunocytochemical analyses were performed in parallel to characterize the nature of the repaired tissue. We show that T-mDPSCs are present up to 3 months postimplantation in the healing defect and that they rapidly differentiate in chondrocyte-like cells expressing all the expected characteristic markers. T-mDPSCs further maturate into hypertrophic chondrocytes and likely signal to host progenitors that form new bone tissue. This demonstrates that implanted T-mDPSCs are able to survive in the defect microenvironment and to participate directly in repair via an endochondral bone ossification-like process. STEM CELLS 2019;37:701-711 SIGNIFICANCE STATEMENTReconstruction of large bone defects in the craniofacial area poses a continued clinical dilemma. Tissue engineering reconstructive strategies using specific scaffolds combined with stem cells endowed with skeletogenic potentials appear as efficient alternatives. The survival of the cells and their direct participation to new bone formation is, however, still a matter of debate. In this study, implanted cell survival in the defect by using fluorescent dental pulp stem cells and their in vivo tracking by two-photon microscopy was demonstrated. Immunohistochemical analyses further reveal that they directly participate in bone repair through an endochondral ossification process. This study paves the way for a better understanding of the molecular and cellular mechanisms at play during (craniofacial) bone repair before their translation to clinical applications.
In humans, the SOST gene encodes sclerostin, an inhibitor of bone growth and remodeling, which also negatively regulates the bone repair process. Sclerostin has also been implicated in tooth formation, but its potential role in pulp healing remains unknown. The aim of this study was to explore the role of sclerostin in reparative dentinogenesis using Sost knockout mice ( Sost). The pulps of the first maxillary molars were mechanically exposed in 3-mo-old Sost and wild-type (WT) mice ( n = 14 mice per group), capped with mineral trioxide aggregate cement, and the cavities were filled with a bonded composite resin. Reparative dentinogenesis was dynamically followed up by micro-computed tomography and characterized by histological analyses. Presurgical analysis revealed a significantly lower pulp volume in Sost mice compared with WT. At 30 and 49 d postsurgery, a large-forming reparative mineralized bridge, associated with osteopontin-positive mineralization foci, was observed in the Sost pulps, whereas a much smaller bridge was detected in WT. At the longer time points, the bridge, which was associated with dentin sialoprotein-positive cells, had expanded in both groups but remained significantly larger in Sost pulps. Sclerostin expression in the healing WT pulps was detected in the cells neighboring the forming dentin bridge. In vitro, mineralization induced by Sost dental pulp cells (DPCs) was also dramatically enhanced when compared with WT DPCs. These observations were associated with an increased Sost expression in WT cells. Taken together, our data show that sclerostin deficiency hastened reparative dentinogenesis after pulp injury, suggesting that the inhibition of sclerostin may constitute a promising therapeutic strategy for improving the healing of damaged pulps.
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